TY - JOUR
T1 - Duplex quantitative Reverse-Transcriptase PCR for simultaneous assessment of drug activity against Leishmania intracellular amastigotes and their host cells
AU - van den Bogaart, Erika
AU - Schoone, Gerard J.
AU - Adams, Emily R.
AU - Schallig, Henk D. F. H.
PY - 2014
Y1 - 2014
N2 - Currently available drugs for treatment of Leishmania infections are highly toxic and drug resistance to first line therapies has been observed. New, safer and more effective drugs are urgently needed to improve clinical resolution of the disease and reduce the risks associated with it. High-throughput screening of new compounds against cultured promastigotes is easy to perform, but the results are poorly predictive of in vivo efficacy. Intra-macrophage amastigote models provide a better proxy of the clinically relevant stage of disease and should be routinely implemented in the search for new anti-leishmanial agents, despite being labor intensive. This study describes the use of a duplex quantitative Reverse-Transcriptase PCR (qRT-PCR) for assessment of drug activity against Leishmania intracellular amastigotes and their host cells. The assay simultaneously quantifies Leishmania 18S ribosomal RNA and the human beta 2-microglobulin (beta-2M) mRNA, used for monitoring drug cytotoxicity and test performance. Accurate determination of parasite viability by the newly developed qRT-PCR was confirmed by parallel assessment of compound performance against standard microscopy. Highly reproducible anti-leishmanial activities were obtained with a set of structurally- and pharmacologically-diverse compounds, whose toxicity against host cells correlated with a low beta-2M amplification. Sensitive and versatile, this duplex qRT-PCR offers a valuable tool for assessment of drug activities against Leishmania amastigotes and their host cells. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved
AB - Currently available drugs for treatment of Leishmania infections are highly toxic and drug resistance to first line therapies has been observed. New, safer and more effective drugs are urgently needed to improve clinical resolution of the disease and reduce the risks associated with it. High-throughput screening of new compounds against cultured promastigotes is easy to perform, but the results are poorly predictive of in vivo efficacy. Intra-macrophage amastigote models provide a better proxy of the clinically relevant stage of disease and should be routinely implemented in the search for new anti-leishmanial agents, despite being labor intensive. This study describes the use of a duplex quantitative Reverse-Transcriptase PCR (qRT-PCR) for assessment of drug activity against Leishmania intracellular amastigotes and their host cells. The assay simultaneously quantifies Leishmania 18S ribosomal RNA and the human beta 2-microglobulin (beta-2M) mRNA, used for monitoring drug cytotoxicity and test performance. Accurate determination of parasite viability by the newly developed qRT-PCR was confirmed by parallel assessment of compound performance against standard microscopy. Highly reproducible anti-leishmanial activities were obtained with a set of structurally- and pharmacologically-diverse compounds, whose toxicity against host cells correlated with a low beta-2M amplification. Sensitive and versatile, this duplex qRT-PCR offers a valuable tool for assessment of drug activities against Leishmania amastigotes and their host cells. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved
U2 - https://doi.org/10.1016/j.ijpddr.2013.11.001
DO - https://doi.org/10.1016/j.ijpddr.2013.11.001
M3 - Article
C2 - 24596664
SN - 2211-3207
VL - 4
SP - 14
EP - 19
JO - INTERNATIONAL JOURNAL FOR PARASITOLOGY-DRUGS AND DRUG RESISTANCE
JF - INTERNATIONAL JOURNAL FOR PARASITOLOGY-DRUGS AND DRUG RESISTANCE
IS - 1
ER -